Specificity and Mode of Action of the Antifungal Fatty Acid cis-9-Heptadecenoic Acid Produced by Pseudozyma flocculosa

cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.     

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.     

Identification of a biosynthesis gene cluster for flocculosin a cellobiose lipid produced by the biocontrol agent Pseudozyma flocculosa

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O‐glycosidically linked to 3,15,16‐trihydroxypalmitic acid. The sugar moiety is acylated with 2‐hydroxy‐octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl‐transferase and is lacking a gene homologous to the α‐hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl‐transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl‐transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.     

Ecological basis of the interaction between Pseudozyma flocculosa and powdery mildew fungi

In this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology of Pseudozyma flocculosa and its biocontrol activity. For this purpose, we compared the development of P. flocculosa to that of a close relative, the plant pathogen Ustilago maydis, under different environmental conditions. This approach was further supported by measuring the expression of cyp1, a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression of cyp1 remained unchanged over time in P. flocculosa and was undetected in U. maydis. At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves, P. flocculosa induced a complete collapse of the pathogen colonies, but glycolipid production, as studied by cyp1 expression, was still comparable to that of controls. In complete contrast, cyp1 was upregulated nine times when P. flocculosa was applied to Botrytis cinerea-infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth of P. flocculosa on powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such as U. maydis do not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity of P. flocculosa toward Erysiphales does appear to be more complex than simple antibiosis.     

Usefulness of heterologous promoters in the Pseudozyma flocculosa gene expression system

The basidiomycetous fungus Pseudozyma flocculosa represents a promising new host for the expression of complex recombinant proteins. Two novel heterologous promoter sequences, the Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase (GPD) and Pseudozyma tsukubaensis alpha-glucosidase promoters, were tested for their ability to provide expression in P. flocculosa. In liquid medium, these two promoters produced lower levels of intracellular green fluorescent protein (GFP) as compared to the U. maydis hsp70 promoter. However, GPD and alpha-glucosidase sequences behaved as constitutive promoters whereas the hsp70 promoter appeared to be morphology-dependent. When using the hsp70 promoter, the expression of GFP increased proportionally to the concentration of hygromycin in the culture medium, indicating possible induction of the promoter by the antibiotic. Optimal solid-state culture conditions were designed for high throughput screening of hygromycin-resistant transformants with the hsp70 promoter in P. flocculosa.     

Polymorphism of linoleic acid (cis-9, cis-12-octadecadienoic acid) and alpha-linolenic acid (cis-9, cis-12, cis-15-octadecatrienoic acid)

Crystallization and polymorphic properties of linoleic acid (cis-9, cis-12-Octadecadienoic acid) (LA) and alpha-linolenic acid (cis-9, cis-12, cis-15-Octadecatrienoic acid) (alpha-LNA) have been studied by optical microscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The DSC analyses presented three polymorphs in LA, and two polymorphs in alpha-LNA. The XRD patterns of the higher- and lower-temperature forms in LA and alpha-LNA showed orthorhombic O'(//)+O-like and O'(//) subcell, which were similar to those of alpha- and gamma-forms of mono-unsaturated fatty acids, respectively. From the solvent crystallization of LA and alpha-LNA in acetonitrile, single crystals of the higher temperature polymorphs have been obtained. The crystal habits of truncated rhombic shape were also similar to those of alpha-forms of the mono-unsaturated fatty acids. The enthalpy and entropy values of fusion and dissolution of the alpha-forms of LA, alpha-LNA and oleic acid showed that the two values decreased with increasing number of the cis-double bond.     

Antifungal activity of extracellular metabolites produced by Sporothrix flocculosa

The antifungal properties of extracellular compounds produced by the epiphytic fungus Sporothrix flocculosa were bioassayed against phytopathogenie fungi on the basis of inhibition of spore germination, and mycelial growth and induction of cellular leakage. Following incubation in stationary culture, S. flocculosa released antifungal metabolites into the culture medium which were extractable with méthylene chloride. When separated by thin layer chromatography, extracted metabolites yielded a compound(s) at R_f0.65 which inhibited development of Cladosporium cucumerinum and several other phytopathogenic fungi. Treatment of Botrytis cinerea and Fusarium oxysporum f.sp. radicis‐lycopersici (FORL) with the same compound(s) greatly reduced spore germination and biomass growth of both fungi. Additionally, both B. cinerea and FORL leaked electrolytes and proteins when grown in presence of the metabolites. Observations under electron microscopy revealed that FORL reacted to the presence of S. flocculosa metabolites by retraction of the plasmalemma and rapid disintegration of the cytoplasm. These reactions were similar to the ones induced by conidia of S. flocculosa when applied on powdery mildew fungi. These results provide strong evidence of the production of antifungal compounds in vivo and of their role in the antagonistic properties of S. flocculosa.     

The mode of action of a nonpolyenic antifungal (desertomycin) produced by a strain of Streptomyces spectabilis

A metabolite with antifungal activity, of non polyenic macrolide structure, was extracted and purified from the culture supernatant of a soil-isolated Streptomyces spectabilis strain, BT 352. This product was found to be related to (or being) desertomycin. Six yeast and five filamentous fungus strains were used to determine minimum concentration of the metabolite that inhibits growth by 80% (IMC); it was established at 50 micrograms/mL for the fungi and at 100 micrograms/mL or more for the yeasts tested. Short-term genotoxicity tests showed no antifungal effect on the bacterial genome, and desertomycin at concentration levels of 100 micrograms/mL or more affected protein synthesis. The antifungal metabolite had no immediate inhibiting effect upon yeast respiration, even at high concentrations; however, the respiration activity of cells grown in the presence of subinhibiting doses and collected during their growth phase was reduced by as much as 40%. Saccharomyces uvarum spheroplast regeneration in a liquid medium containing desertomycin was inhibited at doses fivefold weaker than the IMC determined with intact cells. Contrary to amphotericin B, desertomycin subinhibiting doses do not modify, and if so lightly, the yeast latent phase or the spheroplast wall regeneration phase, thus indicating a fungicidal action. Moreover, following a 30-min contact with desertomycin subinhibiting and inhibiting doses, yeasts liberated potassium in large amounts, indicating that plasma membranes were affected.     

Total synthesis of the novel bacterial fatty acid 16-methyl-8(Z)-heptadecenoic acid.

The recently discovered bacterial fatty acid 16-methyl-8(Z)-heptadecenoic acid was synthesized for the first time in four steps (22% overall yield) starting from commercially available 8-methylnonanoic acid. The synthetic approach provided enough material to corroborate the structure and stereochemistry of the acid, which was recently identified in a Micrococcus bacterium from Lake Pomorie in Bulgaria. Reference equivalent-chain length values in nonpolar capillary gas chromatography for methyl 16-methyl-8(Z)-heptadecenoate and methyl 16-methyl-8(E)-heptadecenoate are also reported. This information will be helpful in subsequent characterizations of these fatty acids, as well as in the total identification of the fatty acid profile of bacteria producing these compounds.     

pH-dependent antifungal lipopeptides and their plausible mode of action

Antimicrobial peptides and lipopeptides play an essential protective role in the innate immune system of all organisms. Despite many studies, the factors that dictate their cell-selectivity and pH-dependent activity are yet not clear. This is important because various organs of the human body have an acidic pH environment, for example, the vagina, gastric lumen, cryogenic dental foci, and lung-lining fluids in cystic fibrosis and asthma. In this study we synthesized a new group of lipopeptides by conjugating dodecanoic acid (DDA) to the N-termini of 12-mer peptides LXXLLXXLLXXL (L(6)X(6), X = Lys, His, Arg, and all the leucines are d-amino acid enantiomers) and investigated their pH-dependent biological activity and a plausible mode of action by using model phospholipids mimicking bacterial, mammalian, and fungal membranes. The data revealed that, depending on the basic amino acid incorporated, the lipopeptides are active against both bacteria and fungi or solely toward fungi. Furthermore, their activity is expressed at an acidic pH alone, neutral pH alone, or at both environments. Determination of secondary structure, membrane leakage experiments, surface plasmon resonance (SPR) binding experiments, and transmission electron microscopy suggest the involvement of a membranolytic effect. This mode of action, which should make it hard for the microorganism to develop resistance, their selective and pH-dependent activity, as well as pharmacological advantages due to the presence of d-amino acids, make them potential candidates for the treatment of mycoses in organs, under various pH environments, especially in cases where the bacterial flora should not be harmed.     

Specificity and mode of action of the muscle-type protein-arginine deiminase.

The primary and secondary specificities and mode of action of the muscle-type protein-arginine deiminase (PAD) were investigated using various derivatives of Arg and its homologues, as well as Arg-containing peptides by quantitative analyses of the reaction products on reverse-phase HPLC. The enzyme converted benzoyl-D-Arg-p-nitroanilide into its citrulline derivative at 18% of the rate of the L-isomer, while the D-Arg residues in peptides were not deiminated to a significant extent. This suggests that PAD does not have strict stereospecificity and it is dependent on the structure of the residues or groups on both sides of the target Arg residue. In contrast, the benzoyl-/-ethyl ester derivatives of homoarginine, alpha-amino-beta-guanidino-propionic acid, canavanine, and NG-methyl-Arg, exhibited poor PAD susceptibility, suggesting that the length and nature of the arm as exactly three CH2 groups, and the integrity of the guanidyl group are quite strict specificity determinants. The enzyme action on Arg residues in peptides depends greatly on their position in the sequence, and on the nature of the neighboring residues. For example, deimination of Arg residues situated at positions 1-3 from the NH2-terminus, except for those preceded by a carbobenzoxy- or benzoyl-group, were in most cases very slow, whereas those at the COOH-terminus were deiminated relatively faster. A single Arg residue sandwiched between two Pro residues was not deiminated at all, while a pair of Arg residues between two Pro were deiminated moderately. Consequently, PAD exhibited a variety of modes of action on more than one Arg residues in the peptides tested. The results suggest the applicability of PAD, albeit quite limited, for selective modification of certain Arg residues in peptides and proteins by appropriately controlling reaction time and several other parameters. The PAD's mode of action was compared with those of three Arg-bond cleaving proteases.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.     

Purification, amino acid sequence and mode of action of bifidocin B produced by Bifidobacterium bifidum NCFB 1454

Bifidocin B produced by Bifidobacterium bifidum NCFB 1454 was purified to homogeneity by a rapid and simple three step purification procedure which included freeze drying, Micro-Cel adsorption/desorption and cation exchange chromatography. The purification resulted in 18% recovery and an approximately 1900-fold increase in the specific activity and purity of bifidocin B. Treatment with bifidocin B caused sensitive cells to lose high amounts of intracellular K+ ions and u.v.-absorbing materials, and to become more permeable to ONPG. Bifidocin B adsorbed to the Gram-positive bacteria but not the Gram-negative bacteria tested. Its adsorption was pH-dependent but not time-dependent. For sensitive cells, the adsorption and lethal action of bifidocin B was very rapid. In 5 min, 95% of bifidocin B adsorbed onto sensitive cells. Several salts inhibited the binding of bifidocin B, which could be overcome by increasing the amount of bifidocin B added. Pre-treatment of sensitive cells and cell walls with detergents, organic solvents or enzymes did not cause a reduction in subsequent cellular binding of bifidocin B, but cell wall preparations treated with methanol:chloroform and hot 20% (w/v) TCA lost the ability to adsorb bifidocin B. Also, the addition of purified heterologous lipoteichoic acid to sensitive cells completely blocked the adsorption of bifidocin B. The amino acid sequence indicated that the bacteriocin contained 36 residues. N-terminal amino acid sequence analysis yielded a sequence of KYYGNGVTCGLHDCRVDRGKATCGIINNGGMWGDIG. Curing experiments with 20 micrograms ml-1 acriflavine yielded cell derivatives that no longer produced bifidocin B but retained immunity to bifidocin B. Production of bifidocin B, but not immunity to bifidocin B, was associated with a plasmid of about 8 kb in this strain.     

Classification and mode of action of membrane-active bacteriocins produced by gram-positive bacteria

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by microorganisms belonging to different eubacterial taxonomic branches. Most of them are small cationic membrane-active compounds that form pores in the target cells, disrupting membrane potentials and causing cell death. The production of small cationic peptides with antibacterial activity is a defense strategy found not only in bacteria, but also in plants and animals. Bacteriocins are classified according to different criteria by different authors; in this review, we will summarize the principal bacteriocin classifications, highlight their main physical and chemical characteristics, and describe the mechanism of some selected bacteriocins that act at the membrane level.     

The mode of action of a morphactin,fluoren-9-carboxylic acid

Fluoren-9-carboxylic acid acts not only as an auxin but also as an gibberellin-antagonist. In the standard pea straight test (S₅ section) for auxin it stimulated elongation, the optimum concentration being 10 mg/l. On the other hand, it inhibited elongation at 0.1 mg/l. This inhibitory effect was more marked when younger tissue (S₁ section) which also responds to gibberellin was used. Interaction of FCA and IAA in the S₅ section has shown that at higher concentration of IAA there seemed to be a suppraoptimal effect, indicating that FCA acted as an auxin. However, in the S₁ section, the stimulating effect of GA₃ was markedly inhibited by 0.1 mg/l FCA; 10 mg/l FCA was either additive or less than additive to GA₃. In the cucumber hypocotyl test FCA itself was inactive up to 100 μg/plant, but it inhibited the GA₃-induced elongation. This inhibition was overcome by increasing the dosage of GA₃. In the same material, the IAA-induced elongation was not affected by FCA. These results indicate that whether FCA acts as an auxin or a gibberellin-antagonist depends on whether the tissue is sensitive to gibberellin and/or auxin.     

GFP technology for the study of biocontrol agents in tritrophic interactions: a case study with Pseudozyma flocculosa

GFP technology was applied to the biocontrol agent (BCA) Pseudozyma flocculosa to study its development and interactions at the tritrophic level plant-powdery mildew-BCA. Transformation experiments with GFP led to the production of a strongly fluorescent strain, Act-4, that displayed biocontrol traits typical of P. flocculosa WT. Following inundative applications, growth of P. flocculosa Act-4 was closely and almost exclusively associated with the colonies of the pathogen regardless of the powdery mildew species or the host plant tested. Development of P. flocculosa Act-4 on control leaves alone was extremely limited 24 h after its application and was typical of the epiphytic growth characterizing this type of yeast-like fungus. Based on the strong correlation between the colonization pattern of the different powdery mildew species tested and the presence of P. flocculosa Act-4, as determined by its fluorescence, it seems that growth of the BCA is dependant on the presence of powdery mildews. These results demonstrate that the GFP technology can be used to study plant-pathogen-BCA interactions and fulfill a wide array of purposes ranging from fundamental observations of the biocontrol behavior of a BCA to very applied ones serving some of the requirements for the registration of BCA's such as defining their environmental fate.